Interferon-gamma producing CD4+ T cells quantified by flow cytometry as early markers for Mycobacterium avium ssp. paratuberculosis infection in cattle.

Current diagnostic methods for Johne's disease in cattle allow reliable detection of infections with Mycobacterium avium ssp. paratuberculosis (MAP) not before animals are 2 years of age. Applying a flow cytometry-based approach (FCA) to quantify a MAP-specific interferon-gamma (IFN-γ) response in T cell subsets, the present study sought to monitor the kinetics of the cell-mediated immune response in experimentally infected calves. Six MAP-negative calves and six calves, orally inoculated with MAP at 10 days of age, were sampled every 4 weeks for 52 weeks post-inoculation (wpi). Peripheral blood mononuclear cells (PBMC) were stimulated with either purified protein derivatives (PPD) or whole cell sonicates derived from MAP (WCSj), M. avium ssp. avium or M. phlei for 6 days followed by labeling of intracellular IFN-γ in CD4+ and CD8+ T cells. No antigen-specific IFN-γ production was detectable in CD8+ cells throughout and the responses of CD4+ cells of MAP-infected and control calves were similar up to 12 wpi. However, the mean fluorescence intensity (MFI) for the detection of IFN-γ in CD4+ cells after WCSj antigen stimulation allowed for a differentiation of animal groups from 16 wpi onwards. This approach had a superior sensitivity (87.8%) and specificity (86.8%) to detect infected animals from 16 wpi onwards, i.e., in an early infection stage, as compared to the IFN-γ release assay (IGRA). Quantification of specific IFN-γ production at the level of individual CD4+ cells may serve, therefore, as a valuable tool to identify MAP-infected juvenile cattle.

First report of paratuberculosis (Johne's disease) in livestock farms of river buffaloes (Bubalus Bubalis) in Nineveh, Iraq.

The present study was designed to investigate Mycobacterium avium subsp. paratuberculosis (MAP) in dairy buffalo herds from six different geographical areas in Nineveh, Iraq. A total of 87 individual faecal samples from river buffaloes, representing 12 dairy herds, were investigated for detection of MAP using cultural, Ziehl­Neelsen and MAP­specific PCR­based methods. Overall, MAP was detected at a higher frequency at herd­level (4/12; 33%) compared to the total individual faecal samples (14/87; 16%) with a cell density ranging from 101 to 103 CFU g­1. A significantly (p < 0.05) higher frequency (9/17; 53%) of MAP was observed in faecal samples collected from clinically diseased as compared to healthy (5/70; 7%) buffaloes selected for the study. However, no statistically significant difference (p ≥ 0.05) was observed in the frequency of MAP occurrence between clinical (9; 64%) and apparently healthy (5; 36%) cases. This report, which is the first MAP study based on data from Iraqi dairy buffalo herds suggests that MAP transmission is a significant health risk for grazing livestock. In conclusion, this study would help farm owners and regulatory authorities to realise the importance of developing and applying best farm management practices in order to prevent transmission of MAP to healthy animals and the environment. In addition, effective diagnostic tests should be taken into account when carrying out the screening tests.

Influence of first colostrum pasteurization on serum immunoglobulin G, iron, and activity of gamma-glutamyltransferase in newborn dairy calves.

BACKGROUND AND AIM: Colostrum pasteurization is an established procedure in dairy farms in developed countries. This practice can improve the health status of the offspring by reducing several pathogens. This study aimed to focus on the pasteurization of bovine first colostrum and its influence on certain important bioactive components. MATERIALS AND METHODS: This study was conducted in Holstein-Friesian bull calves, which were randomly divided into two groups and fed with 6 L of untreated (UT, n=10) or 6 L of heat-treated (HT, 63.5°C for 30 min, n=10) colostrum from their own dam within the first 12 h after birth. Blood samples were taken before, 24 h, and 48 h after first colostrum intake to determine the concentrations of immunoglobulin G (IgG) and iron and the activity of gamma-glutamyltransferase (GGT) in the serum. RESULTS: The level of IgG was not affected by pasteurization (p=0.19). However, a slower increase in GGT activity (p<0.05) and a lower serum iron concentration (p=0.04) were observed in the HT group. CONCLUSION: It can be concluded that pasteurization influences the absorption of colostrum components and therefore, the passive transfer of immunity, although the level of IgG was not affected by pasteurization in this study.

Assessing efficacy of N-Acetyl-l-Cysteine-Sodium Hydroxide on bacterial viability and enhanced recovery of Mycobacterium avium subsp. paratuberculosis from bovine colostrum.

The standard procedure for the improved cultural recovery of viable Mycobacterium spp. from diverse samples mainly depends on reducing the viability of background microbiota using different chemical compounds. This study was designed to i) evaluate the efficacy and comparison between N-Acetyl-l-Cysteine-Sodium hydroxide (NALC-2% NaOH) and hexadecylpyridinium chloride (0.75% HPC) treatment and exposure time on reducing the viability of undesirable microorganisms with minimal impact on colostrum consistency; and ii) assess the impact of NALC-2% NaOH on improved and enhanced recovery of Mycobacterium avium subsp. paratuberculosis (MAP) in spiked postpartum colostrum samples and consistency of colostrum. A total of 40 samples, each treated with NALC-2% NaOH for 15 min or 0.75% HPC for 5 h, were investigated for total mesophilic aerobic bacteria (MAB) and enterobacteria (EB) (CFU mL-1). The results showed that treatment of colostrum samples with NALC-2% NaOH completely eliminated EB and significantly reduced MAB (3.6 log10 CFU mL-1). Conversely, samples treated with 0.75% HPC produced a complex mixture following interaction with the colostrum protein and showed non-significant and variable results. In addition, the spiked colostrum treated with NALC-2% NaOH for 15 min revealed recovery of viable MAP cells with a minimum limit of detection of 1.36 log10 CFU 10 mL-1 where no change in the consistency of colostrum was observed. In conclusion, 15-min NALC-2% NaOH treatment of colostrum may significantly reduce the viability of undesirable microorganisms and help to enhance the efficient recovery of MAP without impacting the consistency of high quality postpartum colostrum. This rapid procedure is suitable for efficient recovery and early detection of MAP as well as preventing its transmission to neonates and young calves in MAP infected herds.

Quantitative assessment of German Holstein dairy cattle colostrum and impact of thermal treatment on quality of colostrum viscosity and immunoglobulins.

OBJECTIVE: This study aimed to determine the color, fat, viscosity, IgG concentration, %Brix and refractive index of fresh postpartum colostrum of German Holstein dairy cattle and assess the impact of different thermal treatments on the visual and dynamic viscosity, in association to IgG concentration, of colostrum that can be used for pasteurization process. RESULTS: Of the total 40 fresh postpartum colostrum, the color of colostrum (ranging from white-pale yellow to yellow and dark-yellowish), fat (1.4-8.2 100 g-1), IgG (4-116 mg mL-1), %Brix (8.5-35.4%), refractive index (1.3454-1.3905 nD), visual (ranging from watery to liquid and thick) and dynamic (4.9-219 cp) viscosity, were recorded. Statistical analysis between visual and dynamic viscosity of fresh colostrum showed significant correlation coefficients (rs = 634). Moreover, a significant correlation between viscosity and three IgG concentrations was also observed. Heat-treated colostrum showed dynamic viscosity ranged from 25 to 3066 cP, where dynamic viscosity of colostrum before- and after heat-treatment showed no significant correlation. Treated colostrum at 60 °C/60 min and 63.5 °C/30 min containing IgG concentration ≤ 80 mg mL-1 and ≤ 68 mg mL-1 showed no significant change in the viscosity and can successfully be applied for pasteurization of first postpartum colostrum.

[Substances in the bovine colostrum - a survey]. / Inhaltsstoffe des bovinen Kolostrums ­ eine Übersicht.

The best studied substances in bovine colostrum are the immunoglobulins. They are absorbed in the small intestine of the neonate by pinocytosis. The Fc-receptor is not highly involved in this process in calves compared to other species. However, this receptor plays a crucial role in the transport of immunoglobulins from the circulation of the dam to the udder and, therefore, into the colostrum. During colostrogenesis, which starts up to 8 weeks prior to parturition, up to 500 g of immunoglobulins are transferred daily by this process. In addition, other components of the bovine colostrum have biological activity. Colostrum-derived growth factors, including IGF-1, EGF and TGF, influence the differentiation of the epithelial cells of the gastrointestinal tract and, therefore, its development. In the udder of the dam, they are involved in various mechanisms of adaption throughout the lactation period. Colostral leucocytes are also transported from the colostrum into the circulation of the offspring, this by a process termed cellular migration. These cells have a specific immunological memory and support the calf in the development of an immune response against specific pathogens the dam was exposed to earlier. Colostral enzymes can be used as an indirect parameter to control for an adequate colostrum supply of the calf (e.g. γ-glutamyltransferase) or have an unspecific antimicrobial potential capacity (e.g. lactate peroxidase, lactoferrin, lysozymes). Vitamins, fats, proteins and mass and trace elements in the colostrum are essential nutrients for the bovine neonate because of the great change in the requirements for the neonatal organism from preto postnatal life. The impact of hormones and other components of the colostrum is still mostly unclear. The composition of the colostrum in the individual cow is influenced by numerous factors, including the number of calvings, the amount of colostrum formed and breed of the dam.

Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd / Cultivo fecal y dos métodos de PCR en materia fecal para el diagnóstico de Mycobacterium avium subsp. paratuberculosis en un hato seropositivo / Cultivo fecal e dois métodos de PCR em matéria fecal para o diagnóstico de Mycobacterium avium subsp. paratuberculosis em um rebanho soropositivo

Abstract Background: paratuberculosis is a slow-developing infectious disease, characterized by chronic granulomatous enterocolitis. This disease has a variable incubation period from 6 months to over 15 years, and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Its detection by direct and indirect diagnostic techniques has been of special interest. Objective: to report the diagnosis and detection of MAP using several diagnostic tests in a herd of the Northern region of Antioquia, Colombia. Methods: serum samples from the study herd were analyzed, using a commercial ELISA (enzyme-linked immunosorbent assay) kit. Fecal samples were cultured by duplicate using Herrold´s egg yolk medium (HEYM), and analyzed by an end- point IS900-specific nested PCR protocol, and a commercial F57-real-time PCR kit. Results: eight out of 27 serum samples in the study herd resulted ELISA-positive. None of fecal samples resulted positive to HEYM culture by duplicate and none were found to be positive by F57-real-time PCR. Seven of the 27 fecal samples were found to be positive by end-point IS900-specific nested PCR. Agreement was found between ELISA and end-point IS900-specific nested PCR in one of the animals. Conclusion: the present study gives information about the agreement between direct and indirect MAP-detection techniques, using different matrixes from animals under the same husbandry conditions.

Resumen Antecedentes: la paratuberculosis es una enfermedad infecciosa de desarrollo lento, caracterizada por una enterocolitis granulomatosa crónica. Esta enfermedad tiene un periodo de incubación que varía entre los 6 meses hasta por más de 15 años, y es causada por Mycobacterium avium subsp. paratuberculosis (MAP). Su detección por técnicas diagnósticas directas e indirectas ha sido de interés especial. Objetivo: reportar el diagnóstico y detección de MAP utilizando varias técnicas diagnósticas en un hato de la región norte de Antioquia, Colombia. Métodos: se analizaron las muestras de suero del hato de estudio utilizando un kit comercial de ELISA (enzyme-linked immunosorbent assay). Las muestras de materia fecal fueron cultivadas por duplicado en Herrold´s egg yolk medium (HEYM), y analizadas mediante un protocolo de PCR anidado específico de IS900 y un kit comercial de PCR en tiempo real para F57. Resultados: ocho de las 27 muestras de suero resultaron positivas por ELISA. Ninguna de las muestras de materia fecal resultó positiva al cultivo en HEYM por duplicado ni por PCR en tiempo real para F57. Siete de las 27 muestras de materia fecal resultaron positivas a PCR anidado específico de IS900. Se encontró concordancia entre el resultado de ELISA y de PCR anidado específico de IS900 en uno de los animales. Conclusión: el presente estudio brinda información acerca de la concordancia entre técnicas directas e indirectas de detección de MAP, utilizando diferentes matrices a partir de animales bajo las mismas condiciones de manejo.

Resumo Antecedentes: a paratuberculosis é uma doença infecciosa de evolução lenta, caracterizada por uma enterocolite granulomatosa crônica. Esta doença tem um período de incubação que varia de 6 meses a 15 anos e é causada pelo Mycobacterium avium subsp. paratuberculosis (MAP). Sua detecção por técnicas de diagnóstico diretos e indiretos tem sido de especial interesse. Objetivo: reportar o diagnóstico e a detecção de MAP utilizando várias técnicas de diagnóstico em um rebanho na região norte de Antióquia, Colômbia. Métodos: foram analisadas amostras de soro do rebanho utilizando um kit comercial de ELISA (enzyme- linked immunosorbent assay). As amostras de fezes foram cultivadas em duplicado em Herrold´s egg yolk medium (HEYM) e analisadas utilizando um protocolo de PCR aninhada específico de IS900 e um kit de PCR em tempo real comercial para F57. Resultados: oito das 27 amostras de soro foram positivas para ELISA. Nenhuma das amostras testadas na cultura de fezes HEYM duplicado foram positivas ou na PCR em tempo real para F57. Sete das 27 amostras de fezes foram positivas na PCR aninhada específica para IS900. Foi encontrada concordância entre o resultado de ELISA e PCR aninhada específica para IS900 em um animal. Conclusão: este estudo fornece informações sobre a correlação entre técnicas de detecção direta e indireta do MAP, utilizando diferentes matrizes de animais sob as mesmas condições de condução.

An intra-laboratory cultural and real-time PCR method comparison and evaluation for the detection of subclinical paratuberculosis in dairy herds.

Mycobacterium avium subsp. paratuberculosis (MAP) is a vigorous microorganism which causes incurable chronic enteritis, Johne's disease (JD) in cattle. A target of control programmes for JD is to accurately detect MAP-infected cattle early to reduce disease transmission. The present study evaluated the efficacy of two different cultural procedures and a TaqMan real-time PCR assay for detection of subclinical paratuberculosis in dairy herds. Therefore, sixty-one faecal samples were collected from two Dutch dairy herds (n = 40 and n = 21, respectively) which were known to be MAP-ELISA positive. All individual samples were assessed using two different cultural protocols in two different laboratories. The first cultural protocol (first laboratory) included a decontamination step with 0.75% hexadecylpyridinium chloride (HPC) followed by inoculation on Herrold's egg yolk media (HEYM). The second protocol (second laboratory) comprised of a decontamination step using 4% NaOH and malachite green-oxalic acid followed by inoculation on two media, HEYM and in parallel on modified Löwenstein-Jensen media (mLJ). For the TaqMan real-time PCR assay, all faecal samples were tested in two different laboratories using TaqMan® MAP (Johne's) reagents (Life Technologies). The cultural procedures revealed positive reactions in 1.64% of the samples for cultivation protocol 1 and 6.56 and 8.20% of the samples for cultivation protocol 2, respectively. The results of the TaqMan real-time PCR performed in two different laboratories yielded 13.11 and 19.76% positive reaction. The kappa test showed proportional agreement 0.54 between the mLJ media (second laboratory) and TaqMan® real-time PCR method (second laboratory). In conclusion, the TaqMan real-time PCR could be a strongly useful and efficient assay for the detection of subclinical paratuberculosis in dairy cattle leading to an improvement in the efficiency of MAP control strategies.

Facts, myths and hypotheses on the zoonotic nature of Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of paratuberculosis (Johne's disease [JD]), a chronic granulomatous enteritis in ruminants. JD is one of the most widespread bacterial diseases of domestic animals with significant economic impact. The histopathological picture of JD resembles that of Crohn's disease (CD), a human chronic inflammatory bowel disease of still unresolved aetiology. An aetiological relevance of MAP for CD has been proposed. This and the ambiguity of other published epidemiological findings raise the question whether MAP represents a zoonotic agent. In this review, we will discuss evidence that MAP has zoonotic capacity.

Genotypes of Mycobacterium avium subsp. paratuberculosis from South American countries determined by two methods based on genomic repetitive sequences.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of Johne's disease or paratuberculosis of ruminants and has been associated with Crohn's disease in humans. In this study, the genotypes of MAP obtained so far in South American countries using a combination of the subtyping methods Mycobacterial Interspersed Repeats Units-Variable Number of Tandem Repeats (MIRU-VNTR) and Multilocus Short Sequence Repeats (MLSSR) were analyzed. Through this analysis, seven different MIRU-VNTR genotypes and seven MLSSR genotypes have been detected. If both methods were combined, nine different genotypes were found. Results revealed the predominance of MIRU-VNTR genotype 1 (INMV 1) and MLSSR genotype A (7 g-10 g-4ggt) among MAP isolates from different host species in South America. These predominant MAP genotypes are also commonly detected in Europe and the United States. This predominance could be the result of higher animal infection ability or better culturability on solid media used for isolation. Further studies on molecular epidemiology of MAP must be carried out in South America to increase our knowledge of the global distribution of MAP.

Induction of matrix metalloproteinases and TLR2 and 6 in murine colon after oral exposure to Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in Crohn's disease. Recent evidence suggests that MAP can induce the expression of Matrix Metalloproteinases (MMPs), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within the present study, we analysed whether oral MAP exposure can induce colonic MMP expression in vivo. In MAP exposed mice MAP and spheroplasts were visualized in intramucosal leukocyte aggregates. MAP exposed mice exhibited a higher colonic expression of Mmp-2, -9, -13, -14, Timp-1, Tlr2, Tlr6, Il-1ß, and Tnf-α. Cell clusters of MMP-9 positive cells adjacent to intramucosal leukocyte aggregates and CD45(+) leukocytes were identified as the major cellular sources of MMP-9. Enhanced TLR2 expression was visualized on the luminal side of colonic enterocytes. Although MAP exposure did not lead to macroscopic intestinal inflammation, the observed MAP spheroplasts in intramucosal leukocyte aggregates together with increased colonic expression of toll-like receptors, pro-inflammatory cytokines, and MMPs upon MAP exposure represents a part of the host immune response towards MAP.

Serological and molecular detection of Mycobacterium avium subsp. paratuberculosis in cattle of dairy herds in Colombia.

The objective of this study is the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by serum enzyme-linked immunosorbent assay (ELISA), fecal polymerase chain reaction (PCR), and fecal culture in Colombian dairy herds. Serum and fecal samples from asymptomatic cows (n = 307) of 14 dairy herds were tested for MAP by an unabsorbed ELISA test (ELISA-A). Serum and fecal samples from positive ELISA-A animals (n = 31) were further tested by an absorbed ELISA test (ELISA-B) and PCR. Fecal samples from animals of herds positive by ELISA-A and PCR (n = 105) were inoculated onto three different culture media. ELISA-A produced positive results in 10% of the serum samples and 71% of the herds. ELISA-B and PCR results were positive in two and six serum and fecal samples from positive ELISA-A animals, respectively. Fecal samples were negative for MAP on all culture media. The results of this study confirmed the presence of MAP in local dairy herds and the difficulties of MAP detection in asymptomatic animals by ELISA, PCR, and fecal culture.

Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in ulcerative colitis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection. METHODS: Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR. RESULTS: MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids. CONCLUSIONS: The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC in vivo. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.

Effect of honey on serum cholesterol and lipid values.

Small studies have suggested that honey benefits patients with high cholesterol concentrations. The present study aimed to confirm this finding in a larger group of subjects. Sixty volunteers with high cholesterol, stratified according to gender and hydroxymethylglutaryl-coenzyme A reductase inhibitor (statin) treatment (yes/no), were randomized to receive 75 g of honey solution or a honey-comparable sugar solution once daily over a period of 14 days. Baseline measurements, including body mass index (BMI) and lipid profile, were obtained, and subjects also completed dietary questionnaires and the Inventory for the Assessment of Negative Bodily Affect-Trait form (INKA-h) questionnaire. Measurements were repeated 2 weeks later. BMI and high-density lipoprotein (HDL) cholesterol values were significantly correlated (r = -0.487; P < .001) as were BMI and a lower ratio of low-density lipoprotein (LDL) cholesterol to HDL cholesterol (r = 0.420; P < .001), meaning that subjects with a high BMI had a lower HDL cholesterol value. INKA-h scores and LDL cholesterol values were also significantly correlated (r = 0.273, P = .042). Neither solution influenced significantly cholesterol or triglyceride values in the total group; in women, however, the LDL cholesterol value increased in the sugar solution subgroup but not in the women taking honey. Although ingesting honey did not reduce LDL cholesterol values in general, women may benefit from substituting honey for sugar in their diet. Reducing the BMI lowers the LDL cholesterol value, and psychological interventions also seem important and merit further investigation.

Evaluation of three molecular methods of repetitive element loci for differentiation of Mycobacterium avium subsp. paratuberculosis (MAP).

The aim of the present study is to evaluate the efficiency of three methods to determine the molecular diversity of 34 Mycobacterium avium subsp. paratuberculosis (MAP) strains isolated from 17 cattle herds. The applied methods included the analysis of sequence polymorphism of the mononucleotide (G1 and G2) and trinucleotide sequences (GGT) of the Short Sequence Repeats (SSR) and the determination of size polymorphism of 9 different Mycobacterial Interspersed Repetitive Units (MIRU) and 6 Variable Number Tandem Repeats (VNTR). Sequence analysis of SSR of 34 isolates showed 4, 6, and 2 alleles of G1, G2, and GGT repeats, respectively. The amplification of the investigated 9 MIRU units revealed only two discriminatory genotyping systems (MIRU2 and MIRU3). Out of 6 VNTR PCR differentiation methods, only one method could be recommended for genotyping purposes. The profile 7g-12g-4ggt-II-b-2 of the combination systems G1-G2-GGT-MIRU2-MIRU3-VNTR1658 dominates among the examined isolates and was detected in 14.7% of the isolates. The use of certain repetitive loci of SSR, MIRU, and VNTR techniques in this study showed greater potential than others for the characterization of MAP isolates. The recommended loci can be used for the epidemiological tracing of MAP field strains and to determine the relationships between isolates in different herds.

Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products.

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.

A comparison of standard cultural methods for the detection of foodborne Salmonella species including three new chromogenic plating media.

In this study the draft of the horizontal method for the detection of Salmonella species from human food and animal feed (ISO 6579:2002) was compared to the European gold standard (DIN EN 12824:1998), including the three new chromogenic plating media AES Salmonella Agar Plate (ASAP), Oxoid Salmonella Chromogen Media (OSCM) and Miller-Mallinson agar (MM). First the growth and appearance of 36 bacterial type strains (Salmonella and other 21 species) on ASAP, OSCM and MM were compared to those on the three traditional agars Brilliant Green Agar according to Edel and Kampelmacher (BGA), Xylose Lysine Deoxycholate Agar (XLD) and Xylose Lysine Tergitol 4 Agar (XLT4). Only on MM agar, did all of 36 tested type strains produce typical colonies, especially strains of S. Senftenberg, Salmonella arizonae, S. Dublin and S. Derby. Artificial inoculation experiments using raw pork ground meat (n=92) were subsequently conducted. A shortened incubation time of 24 h in RVS broth yielded a Salmonella species recovery of 100% from spiked meat samples. Finally, 286 naturally contaminated raw porcine and bovine minced meat samples and raw poultry meat samples were investigated. Forty-three strains from a total of 39 Salmonella-positive samples were found. S. Typhimurium (n=21), with DT 104 L, DT 012 and RDNC being the most prevalent subtypes isolated. D-tartrate-positive S. Paratyphi B (n=2) and S. Saint-Paul (n=3) were also recovered. They were cultured from poultry meat and were multi-resistant against antibiotics including nalidixic acid. Rappaport Vassiliadis broth with soypeptone (RVS) yielded the highest recovery of Salmonella spp. (97,4%) compared to Tetrathionate broth with Novobiocin according to Muller and Kauffman (MKTTn, 94,9%) and Selenite Cystine broth (SC, 38,5%). However, no significant difference was obtained by comparing the ISO 6579:2002 draft to the gold standard.

Detection of central nervous system tissues in meat products: validation and standardization of a real-time PCR-based detection system.

Several phenotypic as well as genotypic methods have been published describing the detection of central nervous system (CNS) tissues that are part of the bovine spongiform encephalopathy (BSE) risk material in food products. However, none of these methods is able to differentiate between CNS tissue of the banned ruminant species and tissues of other animal species. A quantitative and species-specific real-time RT-PCR method has been developed that enables the reliable identification of CNS tissues in meat and meat products. This method is based on a messenger (m)RNA assay that uses bovine, ovine and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as markers. The in-house validation studies evaluated the tissue specificity of up to 15 bovine tissues and the standardization of absolute as well as relative quantitative measurement. The specific amplification of spinal cord and brain tissue GFAP cDNA has been shown previously. In addition, two commercially available ELISA kits were used for the comparative analysis of artificially contaminated minced meat. Small quantities of bovine brain that had been stored over the recommended period of 14 days were examined. The real-time PCR method proved to be suitable for the detection of 0.1% CNS tissue. No false negative results were observed. The quantitative detection of GFAP mRNA using real-time RT-PCR seems a suitable tool in routine diagnostic testing that assesses the illegal use of CNS tissue in meat and meat products. The stability of the selected target region of the GFAP mRNA also allows the detection of CNS tissues after the meat has been processed.

Preliminary study of certain serotypes, genetic and antimicrobial resistance profiles of verotoxigenic Escherichia coli (VTEC) isolated in Bosnia and Germany from cattle or pigs and their products.

The aim of this study was to gather more information on the spread of VTEC serotypes, genetic profiles and resistance patterns from pigs or pork and from cattle or beef in different areas, and to improve detection of the source of outbreaks with a wider data pool. Of 130 Escherichia coli samples isolated from a cattle slaughter house and beef retail products in Sarajevo, Bosnia and Herzegovina (BiH), seven were identified as verotoxigenic (VTEC). In comparison, 22 VTEC of 264 E. coli isolates were isolated from bovine faeces (14) and beef products (8) from Germany. Furthermore 23 VTEC of 76 isolates were identified from pig carcasses (10), faeces (9) and pork products (4) from Germany. Gene detection and serotyping were carried out in our laboratory and in the National Reference Laboratory. Antimicrobial resistance was tested with the dilution method in microtitre plates. All porcine isolates belonged to serotypes thus far not associated with human disease. Bovine VTEC were either serotypes commonly associated with human diseases (O157:H7, O103:H2, O157:H-) or rare serotypes. One serotype (O96:H19) was found only in isolates from Sarajevo. Most German VTEC, especially those of porcine origin, had only vtx2 genes, whereas all Bosnian isolates had vtx1 and vtx2 genes. The eae gene was found only in "classical" VTEC serotypes. All 52 VTEC (100%) investigated were resistant to the three sulfonamides tested; porcine isolates were mainly resistant to oxytetracycline (43%) and chlortetracycline (37%), bovine isolates mainly to trimethoprim/sulfamethoxazole and ampicillin (10% each). If sulfonamide resistances are disregarded, more than half (53.8%) of porcine VTEC were multiresistant and one-fourth (25%) of German bovine isolates, but none of the Bosnian bovine isolates. The results show the considerable spread of resistances in VTEC. These results also point out the necessity of gathering data from different geographical areas in order to be able to identify typical local variations in serotypes or gene expression and thus to trace human infections more quickly to their source.

Collaborative trial for validation of a real-time reverse transcriptase-polymerase chain reaction assay for detection of central nervous system tissues as bovine spongiform encephalopathy risk material: part 1.

A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120 degrees C) and the medium heat-treated samples (sausages cooked at 80 degrees C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.